Field of the Invention
This invention provides methods for toxicological screening of pharmaceuticals and other chemical compounds. The invention specifically provides assays that involve multipotent human stem-like cells (hSLCs), as well as methods for using these cells to detect developmental toxicity or teratogenic effects of pharmaceutical compounds and other chemicals. More particularly, the invention provides an in vitro means for analyzing toxicity of compounds predictive of their toxicity during human development. Candidate predictive biomarkers for toxic or teratogenic effects are also identified and provided herein.
Background Art
Birth defects are a leading cause of infant morbidity and pediatric disorders in the United States, affecting 1 in every 33 infants born (Brent & Beckman, 1990, Bull NY Acad Med 66: 123-63; Rosano et al., 2000, J. Epidemiology Community Health 54:660-66), or approximately 125,000 newborns per year. It is understood that developmental toxicity can cause birth defects, and can generate embryonic lethality, intrauterine growth restriction (IUGR), dysmorphogenesis (such as skeletal malformations), and functional toxicity, which can lead to cognitive disorders such as autism. There is an increasing concern about the role that chemical exposure can play in the onset of these disorders. Indeed, it is estimated that 5% to 10% of all birth defects are caused by in utero exposure to known teratogenic agents which induce developmental abnormalities in the fetus (Beckman & Brent, 1984, Annu Rev Pharmacol 24: 483-500).
Concern exists that chemical exposure may be playing a significant and preventable role in producing birth defects (Claudio et al., 2001, Environm Health Perspect 109: A254-A261). This concern has been difficult to evaluate, however, since the art has lacked a robust and efficient model for testing developmental toxicity for the more than 80,000 chemicals in the market, plus the new 2,000 compounds introduced annually (General Accounting Office (GAO), 1994, Toxic Substances Control Act: Preliminary Observations on Legislative Changes to Make TSCA More Effective, Testimony, Jul. 13, 1994, GAO/T-RCED-94-263). Fewer than 5% of these compounds have been tested for reproductive outcomes and even fewer for developmental toxicity (Environmental Protective Agency (EPA), 1998, Chemical Hazard Data Availability Study, Office of Pollution Prevention and Toxins). Although some attempts have been made to use animal model systems to assess toxicity (Piersma, 2004, Toxicology Letters 149:147-53), inherent differences in the sensitivity of humans in utero have limited the predictive usefulness of such models. Development of a human-based cell model system would have an enormous impact in drug development and risk assessment of chemicals.
Toxicity, particularly developmental toxicity, is also a major obstacle in the progression of compounds through the drug development process. Currently, toxicity testing is conducted on animal models as a means to predict adverse effects of compound exposure, particularly on development and organogenesis in human embryos and fetuses. The most prevalent models that contribute to FDA approval of investigational new drugs are whole animal studies in rabbits and rats (Piersma, 2004, Toxicology Letters 149: 147-53). In vivo studies rely on administration of compounds to pregnant animals at different stages of pregnancy and embryonic/fetal development (first week of gestation, organogenesis stage and full gestation length). However, these in vivo animal models are limited by a lack of biological correlation between animal and human responses to chemical compounds during development due to differences in biochemical pathways. Species differences are often manifested in trends such as dose sensitivity and pharmacokinetic processing of compounds. According to the reported literature, animal models are approximately 60% efficient in predicting human developmental response to compounds (Greaves et al., 2004, Nat Rev Drug Discov 3:226-36). Thus, human-directed predictive in vitro models present an opportunity to reduce the costs of new drug development and enable safer drugs.
In vitro models have been employed in the drug industry for over 20 years (Huuskonen, 2005, Toxicology & Applied Pharm 207:S495-S500). Many of the current in vitro assays involve differentiation models using primary cell cultures or immortalized cells lines (Huuskonen, 2005, Toxicology & Applied Pharm 207:S495-S500). Unfortunately, these models differ significantly from their in vivo counterparts in their ability to accurately assess development toxicity. In particular, the ECVAM initiative (European Center for Validation of Alternative Methods) has used mouse embryonic stem cells as a screening system for predictive developmental toxicology. The embryonic stem cell test (EST) has been able to predict the teratogenicity of 78% of the drugs tested, and the test was reported to be able to differentiate strong teratogens from moderate/weak or non-embryotoxic compounds (Spielmann et al., 1997, In vitro Toxicology 10:119-27). This model is limited in part because toxicological endpoints are defined only for compounds that impair cardiac differentiation. This model also fails to account for interspecies developmental differences between mice and humans, and so does not fully address the need in the art for human-specific model systems.
Thus there remains a need in the art for a human cell derived in vitro method for reliably determining developmental toxicity in pharmaceutical agents and other chemical compounds. There also is a need in the art to better understand human development and its perturbation by toxins and other developmental disrupting agents, to assist clinical management of acquired congenital disorders and the many diseases that share these biochemical pathways, such as cancer. Human derived cell based systems increase the probability of identifying biomarkers of toxicity that may both predict toxicity as well as identify toxicity caused by other diseases.
The association of metabolomics and human embryonic stem cells (hESCs) has led to a more effective in vitro human model to predict developmental toxicity. hESCs were first derived from the inner cell mass of blastocysts (Thomson et al. 1998). Given the human embryonic origin of these cells, an in vitro teratogenicity test using hESCs is likely to produce more accurate human endpoints, while at the same time reducing cost and time and increasing predictability over animal studies. Metabolomics assesses functional changes in biochemical pathways by detecting changes to the dynamic set of small molecules that comprise the metabolome. The feasibility of metabolomics in biomarker discovery has been demonstrated by multiple studies (Cezar et al. 2007, Tan et al. 1998, Sabatine et al. 2005, Barr et al. 2003, Qu et al. 2000).
However, there is an unmet need to develop more accurate methods for human developmental toxicity screening and the establishment of a highly predictive in vitro system for predicting chemical toxicity during early human development.
The present study discloses the establishment of such a system. The present invention further provides for the assessment of a plurality of small molecules, preferably secreted or excreted from human stem-like cells (hSLCs), and is determined and correlated with health and disease or insult state.
The present invention provides a high-throughput developmental toxicity screen that is more predictive than currently available assays and which offers quantitative human endpoints.